Characterization of cells susceptible to SARS-COV-2 and methods for detection of neutralizing antibody by focus forming assay

  1. E. Taylor Stone
  2. Elizabeth Geerling
  3. Tara L. Steffen
  4. Mariah Hassert
  5. Alexandria Dickson
  6. Jacqueline F. Spencer
  7. Karoly Toth
  8. Richard J. DiPaolo
  9. James D. Brien
  10. Amelia K. Pinto

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Abstract

The SARS-CoV-2 outbreak and subsequent COVID-19 pandemic have highlighted the urgent need to determine what cells are susceptible to infection and for assays to detect and quantify SARS-CoV-2. Furthermore, the ongoing efforts for vaccine development have necessitated the development of rapid, high-throughput methods of quantifying infectious SARS-CoV-2, as well as the ability to screen human polyclonal sera samples for neutralizing antibodies against SARS-CoV-2. To this end, our lab has adapted focus forming assays for SARS-CoV-2 using Vero CCL-81 cells, referred to in this text as Vero WHO. Using the focus forming assay as the basis for screening cell susceptibility and to develop a focus reduction neutralization test. We have shown that this assay is a sensitive tool for determining SARS-CoV-2 neutralizing antibody titer in human, non-human primate, and mouse polyclonal sera following SARS-CoV-2 exposure. Additionally, we describe the viral growth kinetics of SARS-CoV-2 in a variety of different immortalized cell lines and demonstrate via human ACE2 and viral spike protein expression that these cell lines can support viral entry and replication.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.20.259838: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Ethics statement: All animal studies were conducted in accordance with the Guide for Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Saint Louis University Animal Care and Use Committee (IACUC; protocol 2771)
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The primary antibody consisted of polyclonal anti-SARS-CoV-2 guinea pig sera (BEI: NR-0361) and was diluted 1:15,000 with FFA Staining Buffer (1 × PBS, 1mg/ml saponin (Sigma: 47036)).
    anti-SARS-CoV-2
    suggested: None
    The secondary antibody consisted of goat anti-mouse conjugated horseradish peroxidase (Sigma: A-7289) diluted 1:5,000 in FFA Staining Buffer.
    anti-mouse
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Virus was passaged two times in Vero E6 cells (ATCC® CRL-1586™) before clarification by centrifugation (3000 rpm for 30 min) and storage at −80°C until further use.
    Vero E6
    suggested: None
    SHHC17 cells were gifted by K. Toth.
    SHHC17
    suggested: None
    Focus forming assay: One day prior to the assay, Vero WHO cells were plated in a 96 well flat bottom tissue culture treated plate.
    Vero WHO
    suggested: ECACC Cat# 88020401, RRID:CVCL_JF53)
    Media was then removed from the 96-well flat bottom plate containing the Vero WHO cell monolayer and replaced with 100μL per well of diluted samples.
    Vero
    suggested: None
    Software and Algorithms
    SentencesResources
    The total peak area under the curve (AUC) was calculated using GraphPad Prism 8.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.08.20.259838v1 on bioRxiv